An antibacterial peptide RF-18 and its application in the preparation of antibacterial products

Abstract

The present invention relates to the technical field of antimicrobial peptides, and particularly to an antimicrobial peptide RF-18 and its application in the preparation of antibacterial products. The amino acid sequence of the antimicrobial peptide RF-18 provided by the present invention is shown in SEQ ID NO.1. The antimicrobial peptide RF-18 provided by the present invention is an antimicrobial peptide that can be artificially synthesized, containing only 18 amino acids, and all amino acids are L-amino acids, which greatly reduces the production cost. In addition, the antimicrobial peptide RF-18 provided by the present invention is a novel antimicrobial peptide with an imperfect amphiphilic structure. This antimicrobial peptide can not only exhibit high and broad-spectrum antibacterial activity, but also has extremely high safety, and has neither cytotoxicity nor hemolytic effect within the concentration range of 800 μg / mL, and can be used for the preparation of antibacterial drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of antimicrobial peptides, and particularly to an antimicrobial peptide RF-18 and its application in the preparation of antibacterial products. Background Art

[0002] The discovery and clinical application of antibiotics are considered to be one of the most important breakthroughs in medicine, and their use has greatly increased human lifespan. However, due to the extensive use or abuse of antibiotics and antibacterial drugs, bacteria have become increasingly resistant to antibiotics, giving rise to a large number of "superbugs", such as extensively drug-resistant Acinetobacter baumannii ( Acinetobacter baumannii ), and Staphylococcus aureus ( Staphylococcus aureus ), etc. In order to address the increasingly serious global problem of antibiotic resistance and avoid the advent of the post-antibiotic era without effective antibiotics, it is urgent to develop new antimicrobial drug molecules.

[0003] Antimicrobial peptides, also known as host defense peptides, are an important part of the body's innate immune system. They can help multicellular organisms such as plants, animals, or humans resist microbial infections and have characteristics such as a fast bactericidal rate and a broad antibacterial spectrum. Different from antibiotics, antimicrobial peptides exert their antibacterial effects mainly by targeting the bacterial cell membrane, which makes it almost impossible for bacteria to develop resistance to antimicrobial peptides. Therefore, antimicrobial peptides can be developed into new antibacterial candidate drugs. However, the sequences of natural antimicrobial peptides are relatively long, and the synthesis cost is relatively high. At the same time, antimicrobial peptides often have poor selectivity and are prone to producing certain toxicity to mammalian cells, which greatly limits the development of antimicrobial peptides as antibacterial candidate drugs. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides an antimicrobial peptide RF-18 and its application in the preparation of antibacterial products. The antimicrobial peptide RF-18 of the present invention is an antimicrobial peptide that can be artificially synthesized, contains only 18 amino acids, and all amino acids are L-amino acids, greatly reducing the production cost; at the same time, the antimicrobial peptide RF-18 of the present invention also has extremely high safety, with no cytotoxicity or hemolytic effect within a concentration range of 800 μg / mL.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides an antimicrobial peptide RF-18, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] Preferably, the C-terminus of the antimicrobial peptide RF-18 is amidated.

[0008] The present invention provides the application of the antibacterial peptide RF-18 described in the above technical solution in the preparation of antibacterial products.

[0009] Preferably, the bacteria are Gram-negative bacteria and / or Gram-positive bacteria.

[0010] Preferably, the Gram-negative bacteria include one or more of Acinetobacter baumannii ( Acinetobacter baumannii ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), and Escherichia coli ( Escherichia coli ); the Gram-positive bacteria include Staphylococcus aureus ( Staphylococcus aureus ).

[0011] Preferably, the antibacterial products include antibacterial preparations or antibacterial drugs.

[0012] The present invention provides an antibacterial drug, the active ingredient of which includes the antibacterial peptide RF-18 described in the above technical solution.

[0013] Preferably, the minimum inhibitory concentration (MIC) of the antibacterial peptide RF-18 in the antibacterial drug is 1.03 - 3.81 μg / mL.

[0014] Preferably, the bacteria are Gram-negative bacteria and / or Gram-positive bacteria.

[0015] Preferably, the Gram-negative bacteria include one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli; the Gram-positive bacteria include Staphylococcus aureus.

[0016] Beneficial effects:

[0017] The present invention provides an antibacterial peptide RF-18, the amino acid sequence of which is as shown in SEQ ID NO.1. The antibacterial peptide RF-18 provided by the present invention is an antibacterial peptide that can be artificially synthesized, only contains 18 amino acids, and all amino acids are L-amino acids, greatly reducing the production cost; in addition, the antibacterial peptide RF-18 provided by the present invention is a novel antibacterial peptide with an imperfect amphiphilic structure. This antibacterial peptide can not only exert high-efficiency and broad-spectrum antibacterial activity, but also has extremely high safety, and has no cytotoxicity or hemolytic effect within a concentration range of 800 μg / mL, and can be used to prepare antibacterial infection drugs. Description of the drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments.

[0019] Figure 1 It is a helical wheel model diagram of the antibacterial peptide RF-18;

[0020] Figure 2 It is the result diagram of the cytotoxicity experiment of the antimicrobial peptide RF-18 on human keratinocytes (HaCaT).

[0021] Figure 3 It is the result diagram of the hemolysis experiment of the antimicrobial peptide RF-18 on human red blood cells. Specific implementation manners

[0022] The present invention provides an antimicrobial peptide RF-18, whose amino acid sequence is shown as SEQ ID NO.1, specifically as follows: RRKVKKVIKAIKKGIKKF. As an implementation manner, the C-terminus of the antimicrobial peptide RF-18 is amidated. The present invention improves the stability of the antimicrobial peptide RF-18 by amidating the C-terminus of the antimicrobial peptide RF-18. As an implementation manner, all amino acids of the antimicrobial peptide RF-18 are of L-type. The present invention uses L-type amino acids as raw materials to synthesize the antimicrobial peptide RF-18, which can greatly reduce the production cost.

[0023] As an implementation manner, the synthesis method of the antimicrobial peptide RF-18 can be the solid-phase peptide synthesis method. The present invention has no special requirements for the solid-phase peptide synthesis method, and the methods well-known to those skilled in the art can be adopted.

[0024] The antimicrobial peptide RF-18 provided by the present invention contains 18 amino acids, with a molecular weight of 2167.83 Daltons and an isoelectric point of 12.06. It is a linear polypeptide, and all amino acids are of L-type. In vitro antibacterial experiments show that RF-18 has broad-spectrum antibacterial activity and shows good antibacterial effects on standard strains and clinically derived strains of Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, and the minimum inhibitory concentration (MIC) is 1.03-3.81 μg / mL. At the same time, RF-18 can form an imperfect amphiphilic α-helix structure, showing extremely high safety, and having neither cytotoxicity nor hemolysis at a concentration range of 800 μg / mL.

[0025] Based on the above advantages, the present invention provides the application of the antimicrobial peptide RF-18 described in the above technical solution in the preparation of antibacterial products. As an implementation manner, the bacteria are Gram-negative bacteria and / or Gram-positive bacteria. As an implementation manner, the Gram-negative bacteria include one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli; the Gram-positive bacteria include Staphylococcus aureus.

[0026] As an implementation manner, the antibacterial product can be a product for inhibiting the growth of bacteria and / or killing bacteria. As an implementation manner, the antibacterial product includes antibacterial preparations or antibacterial drugs. As another implementation manner, the antibacterial drug is an antibacterial infection drug.

[0027] Based on the above advantages, the present invention provides an antibacterial drug, and the active ingredient includes the antibacterial peptide RF-18 described in the above technical solution.

[0028] As an embodiment, the minimum inhibitory concentration of the antibacterial peptide RF-18 in the antibacterial drug is 1.03 - 3.81 μg / mL.

[0029] As an embodiment, the bacteria are Gram-negative bacteria and / or Gram-positive bacteria. As an embodiment, the Gram-negative bacteria include one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli; the Gram-positive bacteria include Staphylococcus aureus.

[0030] As an embodiment, the antibacterial drug further includes pharmaceutically acceptable excipients.

[0031] To further illustrate the present invention, the following describes in detail an antibacterial peptide RF-18 provided by the present invention and its application in the preparation of antibacterial products with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0032] Example 1

[0033] Entrust Girll Biochemicals (Shanghai) Co., Ltd. to synthesize the polypeptide shown in SEQ ID No.1 by solid-phase peptide synthesis method, perform amidation modification at its C-terminus, and finally desalt and purify it by HPLC reverse-phase column chromatography to obtain the antibacterial peptide RF-18.

[0034] Construct the helical wheel model of the antibacterial peptide RF-18 through the website: https: / / heliquest.ipmc.cnrs.fr, and the result is as Figure 1 shown. The result shows that the antibacterial peptide RF-18 exhibits imperfect amphiphilic characteristics.

[0035] Example 2

[0036] Analysis of the antibacterial activity of the antibacterial peptide RF-18 is as follows:

[0037] The test strains are commercially available standard strains and clinically sourced strains. The clinically sourced strains are clinically sourced drug-resistant strains, which are disclosed in Chinese Patent CN116675740A. The specific test strains and their numbers are as follows:

[0038] The test Acinetobacter baumannii strains are: the Acinetobacter baumannii standard strain ATCC19606 and the clinically sourced strains numbered 10769 and 0357;

[0039] The Escherichia coli strains to be tested are: the Escherichia coli standard strain ATCC8739 and the strains with clinical source numbers 0894 and 5017;

[0040] The Pseudomonas aeruginosa strains to be tested are: the Pseudomonas aeruginosa standard strain ATCC27853 and the strains with clinical source numbers 90068 and 17068;

[0041] The Staphylococcus aureus strains to be tested are: the Staphylococcus aureus standard strain ATCC6538 and the strains with clinical source numbers 220823 and 15775.

[0042] Inoculate the strains to be tested onto an LB solid plate. After the colonies grow, pick single colonies and transfer them to an LB liquid medium. Incubate them with shaking at 37°C and 180 rpm for 5 h. Measure the OD of the bacterial solution using an ultraviolet spectrophotometer. 600 According to the ratio of 1 OD 600 = 1×10 9 CFU / mL, dilute the bacterial solution with LB liquid medium to a concentration of 2×10 5 CFU / mL; Add 100 μL of the diluted bacterial solution to each well of a sterile 96-well plate. Then add 100 μL of the test sample diluted in gradient with physiological saline to each well. Mix well with a pipette. After mixing, place it in a 37°C constant temperature incubator and incubate with slow shaking overnight; The test sample is the antimicrobial peptide RF-18; The concentration of the diluted test sample is 0 - 200 μg / mL; After incubation, measure the absorbance of the bacterial solution at OD 600 nm. Take the average value of the sample concentrations in the wells where no bacterial growth is detected and the adjacent wells as the minimum inhibitory concentration (MIC). The results are shown in Table 1.

[0043] Table 1 Minimum inhibitory concentration of antimicrobial peptide RF-18 against the test strains

[0044]

[0045] As can be seen from Table 1, RF-18 showed significant antibacterial effects against both the standard strain of Acinetobacter baumannii (ATCC19606) and clinical isolates (No. 10769 and 0357), with MIC values of 2.64 - 3.81 μg / mL; RF-18 also showed significant antibacterial effects against the standard strain of Escherichia coli (ATCC8739) and clinical isolates (No. 0894 and 5017), with MIC values of 1.03 - 1.76 μg / mL; RF-18 also showed significant antibacterial effects against the standard strain of Pseudomonas aeruginosa (ATCC27853) and clinical isolates (No. 90068 and 17068), with an MIC value of 2.34 μg / mL; RF-18 showed significant antibacterial effects against both the standard strain of Staphylococcus aureus (ATCC6538) and clinical isolates (No. 220823 and 15775), with MIC values of 1.17 - 1.76 μg / mL.

[0046] Example 3

[0047] The cytotoxicity of antibacterial peptide RF-18 was detected as follows:

[0048] When HaCaT cells covered about 80% of the bottom of the culture flask containing DMEM medium, they were first washed 3 times with phosphate buffer, then digested with 0.25% trypsin, and made into a cell suspension with a concentration of 5×10 5 cells / mL with DMEM culture solution. 200 μL of the cell suspension was added to each well of a sterile 96-well plate and cultured overnight; the next day, RF-18 samples with different concentration gradients (0 - 1600 μg / mL) were added respectively, with 3 replicates for each concentration, and cultured for another 24 h; after the culture ended, 15 μL of MTT solution with a concentration of 5 mg / mL was added to each well, and the culture was continued in the dark for 4 h. Subsequently, the culture solution in the plate wells was aspirated, 200 μL of dimethyl sulfoxide (DMSO) was added to each well, the culture plate was placed on a shaker and gently shaken for 10 min to dissolve the crystals, and then the absorbance value of each well was measured at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. According to the absorption value detected at 450 nm, the cell viability in the absence of the sample to be tested was defined as 100%, the relative cell viability in the presence of the sample to be tested was calculated, and a bar chart of the relative cell viability corresponding to the RF-18 sample solution was drawn using GraphPad prism software. The results are shown in Figure 2 .

[0049] As Figure 2 can be seen, RF-18 was not toxic to human keratinocyte HaCaT within the concentration range of 800 μg / mL, and only showed weak cytotoxicity at a concentration of 1600 μg / mL.

[0050] Example 4

[0051] Hemolysis test of antibacterial peptide RF-18 is as follows:

[0052] Mix fresh human whole blood and Alsever's solution at a volume ratio of 1:1, centrifuge at 1000 rpm for 5 min, discard the supernatant, wash the red blood cells with physiological saline, and repeat 3 times until the supernatant no longer shows red, to obtain well-washed red blood cells;

[0053] Dissolve the antibacterial peptide RF-18 prepared in Example 1 in physiological saline, and prepare RF-18 samples to be tested with different concentrations (3.125 - 1600 μg / mL);

[0054] Dilute the well-washed red blood cells with physiological saline to a density of 1×10 7 cells / mL. Incubate the diluted red blood cell suspension with the RF-18 samples to be tested with different concentrations at 37°C for 30 min, then centrifuge at 1000 rpm for 5 min, and measure the absorbance of the supernatant at 540 nm. The positive control uses the same volume of Triton X-100 solution (denoted as PC), and the volume concentration of Triton X-100 in the Triton X-100 solution is 10%; the negative control uses the same volume of physiological saline. Calculate the hemolysis rate of the RF-18 samples to be tested with different concentrations. The calculation method of the hemolysis rate is shown in Formula Ⅰ:

[0055] Formula Ⅰ;

[0056] where A is the OD 540 value of the positive control, B is the OD 540 value of the negative control, and C is the OD 540 value of the sample group to be tested.

[0057] The hemolysis rate results are shown in Figure 3 . It can be seen from Figure 3 that the antibacterial peptide RF-18 shows extremely high safety to human red blood cells, and only shows weak hemolysis at a concentration of 1600 μg / mL (hemolysis rate is 9%). There is no hemolysis effect within the concentration range of 800 μg / mL.

[0058] In summary, the antibacterial peptide RF-18 provided by the present invention is a novel antibacterial peptide with an imperfect amphiphilic structure. This antibacterial peptide can not only exert high and broad-spectrum antibacterial activities, but also has extremely high safety. It has neither cytotoxicity nor hemolysis effect within the concentration range of 800 μg / mL, and can be used to prepare antibacterial infection drugs.

[0059] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antibacterial peptide RF-18, characterized in that, The amino acid sequence is as shown in SEQ ID NO. 1; The C-terminus of the antibacterial peptide RF-18 is amidated.

2. The use of the antibacterial peptide RF-18 according to claim 1 in the preparation of antibacterial products; The bacteria are Gram-negative bacteria and / or Gram-positive bacteria; The Gram-negative bacteria is Acinetobacter baumannii Acinetobacter baumannii , Pseudomonas aeruginosa Pseudomonas aeruginosa and Escherichia coli Escherichia coli ; the Gram-positive bacteria is Staphylococcus aureus Staphylococcus aureus .

3. The application according to claim 2, characterized in that, The antibacterial products include antibacterial preparations or antibacterial drugs.

4. An antibacterial drug, characterized in that, The active ingredient includes the antibacterial peptide RF-18 according to claim 1.

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